Regioselective alkane hydroxylation with a mutant CYP153A6 enzyme

ABSTRACT

Cytochrome P450 CYP153A6 from  Myobacterium  sp. strain HXN1500 was engineered using in-vivo directed evolution to hydroxylate small-chain alkanes regioselectively. Mutant CYP153A6-BMO1 selectively hydroxylates butane and pentane at the terminal carbon to form 1-butanol and 1-pentanol, respectively, at rates greater than wild-type CYP153A6 enzymes. This biocatalyst is highly active for small-chain alkane substrates and the regioselectivity is retained in whole-cell biotransformations.

GOVERNMENT INTERESTS

The United States Government has rights in this invention pursuant toContract No. DE-FG02-06ER15762, between the U.S. Department of Energy(DOE) and the California Institute of Technology.

REFERENCE TO SEQUENCE LISTING

The electronic readable copy and paper copy of the sequence listings forthis invention are identical.

FIELD OF THE INVENTION

One or more embodiments of the present invention are concerned withvariants of novel, mutant CYP153A6 enzymes that display altered andimproved regioselectivity in their selective hydroxylation ofsmall-chain alkanes. One or more embodiments also relate to novelvariants of CYP153A6 enzymes that are capable of hydroxylating butanesand smaller alkanes at the terminal position at a rate greater thanwild-type CYP153A6 enzymes. One or more embodiments also relate to amethod of altering the ability of a CYP153A6 enzyme to selectivelyhydroxylate small-chain alkanes at the terminal position.

BACKGROUND

Microbial utilization and degradation of alkanes was discovered almost acentury ago. Since then, several enzyme families capable ofhydroxylating alkanes to alkanols, the first step in alkane degradation,have been identified and categorized based on their preferredsubstrates. The soluble and particulate methane monooxygenases (sMMO andpMMO) and the related propane monooxygenase and butane monooxygenases(BMO) are specialized on gaseous small-chain alkanes (C₁ to C₄), whilemedium-chain (C₅ to C₁₆) alkane hydroxylation appears to be the domainof the CYP153A6 and AlkB enzyme families.

Conversion of C₁ to C₄ alkanes to the corresponding alkanols is ofparticular interest for producing liquid fuels or chemical precursorsfrom natural gas. The MMO-like enzymes that catalyze this reaction innature, however, exhibit limited stability or poor heterologousexpression and have not been suitable for use in a recombinant host thatcan be engineered to optimize substrate or cofactor delivery. (vanBeilen, J. B., et. al., 2007, Appl. Microbiol. Biotechnol., 74, 13-21).Alkane monooxygenases often cometabolize a wider range of alkanes thanthose which support growth.

CYP153 is a family of enzymes that has recently been shown tohydroxylate alkanes to the corresponding alkanols. (Maier, T. H., et.al., 2001, Biochem. Biophys. Res. Commun., 286, 652-658; Funhoff, E. G.,et. al., 2006, J. Bacteriol., 188, 5220-5227; Funhoff, E. G., et. al.,2007, Enzyme Microb. Technol., 40, 806-812; and, van Beilen, J. B., et.al. 2006, Appl. Environ. Microb., 72, 59-65). (16, 9, 10, 31). Thisfamily of heme-containing cytochrome P450 monooxygenases has been thesubject of biochemical studies and known substrates includes alkanescontaining five to eleven carbons. (Müller, R., et. al., 1989, Biomed.Biochim. Acta, 48, 243-254; Maier, T. H., et. al., 2001, Biochem.Biophys. Res. Commun., 286, 652-658; and, Funhoff, E. G., et. al., 2006,J. Bacteriol., 188, 5220-5227). (9,16,19). The best characterized memberof this family, CYP153A6, hydroxylates it's preferred substrate, octane,predominantly at the terminal position. (Müller, R., et. al., 1989,Biomed. Biochim. Acta, 48, 243-254; Funhoff, E. G., et. al., 2007,Enzyme Microb. Technol., 40, 806-812; and, Kubota, M., et. al., 2005,Biosci. Biotechnol. Biochem., 69, 2421-2430). (9,10,14). Nucleotide andamino acid sequences for CYP153A6 from Myobacterium sp. HXN-1500 can befound in, and are hereby incorporated by reference from, the GenBankdatabase under the accession Nos. AJ783967 (SEQ ID NO: 1) and Q65A64(SEQ ID NO: 2), respectively. However, the CYP153A6 gene used as aparent gene in this study was found to lack the first three codonscompared to the AJ783967 sequence, probably due to cloning procedures.However, the encoded protein was otherwise identical, fully functionaland performed identical to the Q65A64 protein under all analyzedconditions. Therefore, it will also be referred to as an amino acidhaving the sequence set forth in SEQ ID NO: 3.

The soluble class II-type three-component CYP153A6 enzymes are the mainactors in medium-chain length alkane hydroxylation by the cultivatedbacteria to date. (van Beilen, J. B., et. al. 2006, Appl. Environ.Microb., 72, 59-65). Recent studies have shown that high activities onsmall alkanes can be obtained by engineering bacterial P450 enzymes suchas P450cam (CYP101, a camphor hydroxylase) and P450 BM3 (CYP102A, afatty acid hydroxylase) (Fasan, R., et. al., 2007, Angew. Chem. Int. Ed.Engl., 46, 8414-8418; Xu, F. et. al., 2005, Angew. Chem. Int. Ed., 44,4029-4032). However, the resulting enzymes hydroxylate propane andhigher alkanes primarily at the more energetically favorable subterminalpositions. Highly selective and desirable terminal hydroxylation isdifficult to achieve by engineering a subterminal hydroxylase. (Peters,M., et. al., 2003, J. Am. Chem. Soc., 125, 13442-13450).

Previous approaches, some of which are described above, relate to invitro evolution of hydroxylase enzymes that does not provide theopportunity to screen for improved activity on a specific alkanesubstrate directly. Further, the prior methods lead to low or noterminal hydroxylation activity and often result in high uncoupling.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the multipleembodiments of the present invention will become better understood withreference to the following description, appended claims, and accompanieddrawings where:

FIG. 1 is a graphical representation of the growth of P. putidaGPo12(pGEc47ΔB) with primary and secondary linear short and medium-chainlength alkanes as measured by the optical density of the cultures after18 days of growth in liquid M9 minimal medium with 0.5% (vol/vol)primary or secondary alcohols as the carbon source dissolved in 5%(vol/vol) organic layer of heptamethylnonane.

FIG. 2 is a schematic representation outlining the genesis of strainPcyp2 pCom8*_cyp153A6-BMO1.

FIG. 3 represents the bioconversion of alkanes to 1- and 2-alkanols byCYP153A6 variants, wherein the white graph depicts wild-type activityand light gray shows CYP153A6-BMO1 activity. The lower portion of eachgraph represents relative activities for 1-alkanol, while the upper partof each graph shows 2-alkanol production.

FIG. 4 is a graphical representation of the CO difference UV-VIS spectraof lysed E. coli BL21(DE3) cell suspensions expressing CYP153A6 orCYP153A6-BMO1 concentrated in 5:1 buffer, and a negative controlcarrying an empty vector.

FIG. 5 represents the stability of expressed P450 in cell-free extractsexpressing CYP153A6 wild-type (diamonds) and CYP1553A6-BMO1 (squares).

DETAILED DESCRIPTION OF THE INVENTION

One or more embodiments of the invention include a novel mutant CYP153A6enzyme, wherein said mutant enzyme hydroxylates small-chain alkanes atsignificantly higher levels than that of the wild-type CYP153A6 enzyme.

A salient aspect of the one or more embodiments of the present inventionis the surprising ability of the invented CYP153A6 mutant enzyme tohydroxylate small-chain alkanes at significantly higher levels than thatof the wild-type CYP153A6 enzyme, as depicted in Tables 1 and 2 and FIG.3.

Another salient aspect of the one or more embodiments of the presentinvention is the ability of the mutant CYP153A6 enzyme to selectivelyhydroxylate small-chain alkanes at the terminal position. This findingis both surprising and unexpected since the wild-type CYP153A6 enzymeexhibits a preference for alkane substrates having greater than fivecarbon atoms. However, the mutant CYP153A6-BMO1 enzyme exhibited a 75%higher reactive activity towards 1-butanol production than wild-typeCYP153A6. Additionally, it is also surprising and unexpected that themutant CYP153A6-BMO1 exhibits increased selectivity for terminalhydroxylation of butane when compared to the wild-type CYP153A6, asshown in Table 2 and FIG. 3. More specifically, the mutant CYP153A6-BMO1enzyme surprisingly increased the selectivity for terminal hydroxylationof butanol from 78% to 89% of total alkanol product.

In one embodiment, mutants of CYP153A6 from Myobacterium sp. HXN-1500were engineered using directed evolution, as discussed more completelybelow. The initial wild-type CYP153A6 enzyme allowed it to hydroxylatemedium-chain alkanes to produce certain amounts of particularregiospecific alkane products—i.e., 1-alkanols. This wild-type enzymewas then engineered, in vivo, to support bacterial growth on short-chainalkanes while maintaining its regioselectiveness for terminalhydroxylation. The mutant CYP153A6-BMO1 enzyme surprisingly exhibited anincreased production of 1-alkanol when compared to the wild-typeCYP153A6 enzyme.

Additional embodiments of the invention include mutant CYP153A6 enzymeswith altered regioselectivity that are capable of supporting growth ofan expression host on short-chain alkanes. For example, one mutantenzyme, CYP153A6-BMO1, was found to hydroxylate butane at the terminalposition to form 1-butanol at a significantly greater rate thanwild-type CYP153A6.

Additional embodiments include an isolated nucleic acid molecule of SEQID NO: 1 that encodes a polypeptide comprising the amino acid sequenceof SEQ ID NO: 2, having an amino acid substitution corresponding toA97V.

A salient aspect of one or more embodiments of the preferred embodimentsis a point mutation corresponding to an amino acid substitution of A97Vin SEQ ID NO: 2. Experimental evidence suggests that this point mutationdramatically increases the ability of the CYP153A6 enzyme to hydroxylatesmall-chain alkanes at the terminal position. (See FIG. 3).

One preferred embodiment of the present invention employs a contiguousspan of amino acids, wherein the span is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acids of SEQ IDNO: 2, wherein the fragment has an amino acid substitution correspondingto A97V.

Another preferred embodiment employs a polypeptide fragment consistingessentially of SEQ ID NO: 2, wherein the fragment has an amino acidsubstitution corresponding to A97V.

Another embodiment includes an isolated polypeptide or fragment thereofhaving at least 90% identity, and preferably 95% identity, with theamino acid sequence of SEQ ID NO: 2 and having an amino acidsubstitution corresponding to A97V.

Another embodiment includes an isolated polypeptide comprising the aminoacid sequence of SEQ ID NO: 3, having an amino acid substitutioncorresponding to A94V.

A salient aspect of one or more embodiments of the preferred embodimentsis a point mutation corresponding to an amino acid substitution of A94Vin SEQ ID NO: 3. Experimental evidence suggests that this point mutationdramatically increases the ability of the CYP153A6 enzyme to hydroxylatesmall-chain alkanes at the terminal position. (See FIG. 3).

One preferred embodiment of the present invention employs a contiguousspan of amino acids, wherein the span is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acids of SEQ IDNO: 3, wherein the fragment has an amino acid substitution correspondingto A94V.

Another preferred embodiment employs a polypeptide fragment consistingessentially of SEQ ID NO: 3, wherein the fragment has an amino acidsubstitution corresponding to A94V.

Another embodiment includes an isolated polypeptide or fragment thereofhaving at least 90% identity, and preferably 95% identity, with theamino acid sequence of SEQ ID NO: 3, and having a point mutationcorresponding to an amino acid substitution of A94V.

Yet another embodiment includes a method of in vivo directed evolutionto improve the ability of CYP153A6 enzyme to hydroxylate small-chainalkanes at the terminal position.

In another embodiment, an isolated nucleic acid encoding a CYP153A6enzyme that has a higher capability than the corresponding wild-typeCYP153A6 enzyme to oxidize at least one substrate at the terminalposition selected from an alkane comprising carbon-chain of no more thanfive carbons is provided.

Yet another embodiment includes the method of using the above-describedmutants for the selective hydroxylation of short-chain alkanes toproduce well-characterized products in known quantities. As the mutantenzymes produce known products in a known amount, all that is requiredto create a desired product is to select an appropriate mutant CYP153A6enzyme that catalyzes a reaction to produce a desired regiospecificproduct and then apply the substrate to the enzyme under conditionswhich allow for catalysis. Methods of selecting and isolating thedesired product from the products created are also known and disclosedherein.

The invention also contemplates certain modifications to the sequencesdescribed above with codons that encode amino acids that are chemicallyequivalent to the amino acids in the native protein. An amino acidsubstitution involving the substitution of amino acid with a chemicallyequivalent amino acid is known as a conserved amino acid substitution.

Chemical equivalency can be determined by one or more the followingcharacteristics: charge, size, hydrophobicity/hydrophilicity,cyclic/non-cyclic, aromatic/non-aromatic etc. For example, a codonencoding a neutral non-polar amino acid can be substituted with anothercodon that encodes a neutral non-polar amino acid, with a reasonableexpectation of producing a biologically equivalent protein. Amino acidscan generally be classified into four groups. Acidic residues arehydrophillic and have a negative charge to loss of H+ at physiologicalpH. Basic residues are also hydrophillic but have a positive charge toassociation with H+ at physiological pH. Neutral nonpolar residues arehydrophobic and are not charged at physiological pH. Neutral polarresidues are hydrophillic and are not charged at physiological pH. Aminoacid residues can be further classified as cyclic or noncyclic andaromatic or nonaromatic, self-explanatory classifications with respectto side chain substituent groups of the residues, and as small or large.The residue is considered small if it contains a total of 4 carbon atomsor less, inclusive of the carboxyl carbon. Small residues are alwaysnon-aromatic. Of naturally occurring amino acids, aspartic acid andglutamic acid are acidic; arginine and lysine are basic and noncylclic;histidine is basic and cyclic; glycine, serine and cysteine are neutral,polar and small; alanine is neutral, nonpolar and small; threonine,asparagine and glutamine are neutral, polar, large and nonaromatic;tyrosine is neutral, polar, large and aromatic; valine, isoleucine,leucine and methionine are neutral, nonpolar, large and nonaromatic; andphenylalanine and tryptophan are neutral, nonpolar, large and aromatic.Proline, although technically neutral, nonpolar, large, cyclic andnonaromatic is a special case due to its known effects on secondaryconformation of peptide chains, and is not, therefore included in thisdefined group.

There are also common amino acids which are not encoded by the geneticcode included by example and not limitation: sarcosine, beta-alanine,2,3 diamino propionic and alpha-aminisobutryric acid which are neutral,nonpolar and small; t-butylalanine, t-butylglycine, β-methylisoleucine,norleucine and cyclohexylalanine which are neutral, nonpolar, large andnonaromatic; ornithine which is basic and noncyclic; cysteic acid whichis acidic; citrulline aceyl lysine and methionine sulfoxide which areneutral, polar, large and nonaromatic; and phenylglycine,2-napthylalanine, β-thienylalanine and 1,2,3,4, tetrahydroisoquinoline-3carboxylic acid which are neutral, nonpolar, large and aromatic. Othermodifications are known in the art some of which are discussed in U.S.Pat. No. 6,465,237 issued to Tomlinson on Oct. 15, 2002, which isincorporated herein by reference.

I. DEFINITIONS

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton et. al.(2001) Dictionary of Microbiology and Molecular Biology, third edition,John Wiley and Sons (New York); and, Hale and Marham (1991) The HarperCollins Dictionary of Biology, Harper Perennial, N.Y. provide one ofskill with a general dictionary of many of the terms used in thisinvention. In practicing one or more embodiments of the presentinvention, several conventional techniques in molecular biology,proteomics, microbiology and recombinant DNA are used. Such techniquesare well known and are explained in, for example, Sambrook, 1999,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: Apractical Approach, 1985 (D. N. Glover ed.); Current Protocols inMolecular Biology, John Wiley & Sons, Inc. (1994); Proteins andProteomics: A Laboratory Manual (Simpson ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2002),www.proeteinsandproteomics.org, and all more recent editions of thesepublications. Although any methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, the preferred methods and materials are described.For purposes of the present invention, the following terms are definedbelow.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

As used herein, “about” or “approximately” shall mean within 20 percent,preferably within 10 percent, and more preferably within 5 percent of agiven value or range.

The term “identical” in the context of two nucleic acid or polypeptidesequences refers to the residues in the two sequences which are the samewhen aligned for maximum correspondence over a specified comparisonwindow. When percentage of sequence identity is used in reference toproteins or peptides it is recognized that residue positions which arenot identical often differ by conservative amino acid substitutions,where amino acid residues are substituted for other amino acid residueswith similar chemical properties (e.g. charge or hydrophobicity) andtherefore do not change the functional properties of the molecule. Wheresequences differ in conservative substitutions, the percent sequenceidentity may be adjusted upwards to correct for the conservative natureof the substitution. Means for making this adjustment are well-known tothose of skill in the art. Typically this involves scoring aconservative substitution as a partial rather than a full mismatch,thereby increasing the percentage sequence identity. Thus, for example,where an identical amino acid is given a score of 1 and anon-conservative substitution is given a score of zero, a conservativesubstitution is given a score between zero and 1. The scoring ofconservative substitutions is calculated, e.g., according to thealgorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17(1988) e.g., as implemented in the program PC/GENE (Intelligenetics,Mountain View, Calif., USA).

The term “substrate” means any substance or compound that is convertedor meant to be converted into another compound by the action of anenzyme catalyst. The term includes aromatic and aliphatic compounds, andincludes not only a single compound, but also combinations of compounds,such as solutions, mixtures and other materials which contain at leastone substrate.

An “oxidation reaction” or “oxygenation reaction”, as used herein, is achemical or biochemical reaction involving the addition of oxygen to asubstrate, to form an oxygenated or oxidized substrate or product. Anoxidation reaction is typically accompanied by a reduction reaction(hence the term “redox” reaction, for oxidation and reduction). Acompound is “oxidized” when it receives oxygen or loses electrons. Acompound is “reduced” when it loses oxygen or gains electrons.

The term “enzyme” means any substance composed wholly or largely ofprotein or polypeptides that catalyzes or promotes, more or lessspecifically, one or more chemical or biochemical reactions.

A “polypeptide” (one or more peptides) is a chain of chemical buildingblocks called amino acids that are linked together by chemical bondscalled peptide bonds. A protein or polypeptide, including an enzyme, maybe “native” or “wild-type”, meaning that it occurs in nature or has theamino acid sequence of a native protein, respectively. These terms aresometimes used interchangeably. A polypeptide may or may not beglycosylated.

A “recombinant wild-type” typically means the wild type sequence in arecombinant host without glycosylation. Comparisons in the examples andfigures of this application are generally with reference to a wild typethat is a recombinant wild type.

A polypeptide may also be a “mutant,” “variant” or “modified”, meaningthat it has been made, altered, derived, or is in some way different orchanged from a native protein or its wild-type composition, or fromanother mutant. Mutant proteins typically have amino acid substitutionsat one or more positions. Mutant DNA molecules typically have nucleotidesubstitutions in one or more positions. Mutant forms of a protein or DNAmolecule can have the same, or altered, functions in comparison to thewild-type. For ease of discussion, mutants may be referred to by theirvariation from the single amino acid code from which the mutation arose.For example, in one format the mutant is referred to as XPOSY, where “X”refers to the single letter code of the amino acid in the originalsequence, “POS” refers to the position of the mutation in the sequence,and Y refers to the single letter code for the new amino acid appearingat the mutation's position. For example, V1751 would mean that in theoriginal protein, the amino acid at position 175 is a valine (“V”), butin the mutant, the valine is replaced with an isoleucine (“I”).

A “parent” polypeptide or enzyme is any polypeptide or enzyme from whichany other polypeptide or enzyme is derived or made, using any methods,tools or techniques, and whether or not the parent is itself a native ormutant polypeptide or enzyme. A parent polynucleotide is one thatencodes a parent polypeptide.

As used herein, a “core mutation” is a mutation of a wild-type CYP153A6enzyme that provides the protein with enhanced alkane hydroxylaseactivity. It should be realized that any mutation, or set of mutations,that enhance the ability of a CYP153A6 protein to hydroxylate alkanesare considered core mutations.

A “core mutant” is a CYP153A6 protein that has been altered to containone or more core mutations. In one embodiment, a core mutant is thecytochrome CYP153A6-BMO1 protein which was derived from mutations ofCYP153A6, and includes a A97V core mutation. In one embodiment, thosemutations that revert the amino acid sequence back to the wild typesequence for the selective hydroxylation mutations are not consideredcore mutations.

As used herein, the terms “selective hydroxylation mutations” or“selective mutations” are used interchangeably and refer to mutationsthat provide a protein with altered regio-selectivity towards specificsubstrates. A protein having such mutations is termed a “selectivehydroxylation mutant” or a “selective mutant”. In one embodiment, thetarget substrate of such mutants is a small-chain alkane. The selectivehydroxylation mutations may simply alter the selectivity of the proteintowards a single substrate, or across many substrates. The selectivemutation may alter both the selectivity and increase the functionalability of the enzyme, so that more regio-selective end product isproduced.

Non-limiting general examples of selective hydroxylation mutants showingaltered or enhanced regioselective hydroxylation include CYP153A6-BMO1proteins having one or more of the following additional mutations: A97Vin SEQ ID NO: 2 and A94 V in SEQ ID NO: 3.

In some embodiments, more than a single mutation may be required inorder for the desired result to occur, in such situations, each of therequired mutations will be considered as either core, selective, orboth, as appropriate.

An enzyme is “regioselective” if the product that results from theenzymatic reaction is positioned in an altered or specified position. Inone embodiment, the enzyme is an alkane hydroxylase and thehydroxylation reaction results in a hydroxyl group positioned at theterminal position. This means that while the original enzyme may havecreated a first amount of product A and a second amount of product B,the regioselective enzyme could produce a third amount of product A anda fourth amount of product B. Thus, while the initial wild-type parentcould be considered regioselective for particular substrates, theregioselective mutants described herein display differentregioselectivity from the wild-type parent enzyme. In one embodiment, adistribution of hydroxyl groups in the final product that differs fromthe product of the wild-type enzyme is sufficient to demonstrate thatthe enzyme is regioselective. In another embodiment, an increase of 1,1-2, 2-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90,90-100, 100-200, 200-500 percent or more in the concentration of oneproduct over another product is sufficient to demonstrate that theenzyme is regioselective. In one embodiment, an enzyme is regioselectivewhen its selectivity is greater than the wild-type enzyme as shown inTable 2 and FIG. 3.

A “consistent” selective mutant is a selective mutant that displays aconsistent bias of selectivity of product produced for more than onestarting substrate. Thus, for example, the mutant CYP153A6-BMO1,discussed below, is a consistent regioselective mutant for propane,butane, pentane and octane, as the products from propane, butane,pentane and octane all result predominantly in the 1-alcohol. In oneembodiment, a mutant CYP153A6 is a consistent regioselective enzyme ifthe largest amount of product produced from propane, butane, pentane andoctane is the 1-alcohol. In one embodiment, the majority of each of theproducts is made at the same position.

An alkane is typically defined as a non-aromatic saturated hydrocarbonwith the sequence of C_(n)H(2_(n)+2).

A “short-chain alkane” is defined as any alkane having less than sixcarbon atoms, e.g., methane, ethane, propane, butane or pentane.

The proteins of the present invention further include “conservativeamino acid substitution variants” (i.e., conservative) of the proteinsherein described. As used herein, a conservative variant refers to atleast one alteration in the amino acid sequence that does not adverselyaffect the biological functions of the protein. A substitution,insertion or deletion is said to adversely affect the protein when thealtered sequence prevents or disrupts a biological function associatedwith the protein. For example, the overall charge, structure orhydrophobic-hydrophilic properties of the protein can be altered withoutadversely affecting a biological activity. Accordingly, the amino acidsequence can often be altered, for example to render the peptide morehydrophobic or hydrophilic, without adversely affecting the biologicalactivities of the protein.

The “activity” of an enzyme is a measure of its ability to catalyze areaction, and may be expressed as the rate at which the product of thereaction is produced. For example, enzyme activity can be represented asthe amount of product produced per unit of time, per unit (e.g.concentration or weight) of enzyme.

The “stability” of an enzyme means its ability to function, over time,in a particular environment or under particular conditions. One way toevaluate stability is to assess its ability to resist a loss of activityover time, under given conditions. Enzyme stability can also beevaluated in other ways, for example, by determining the relative degreeto which the enzyme is in a folded or unfolded state. Thus, one enzymeis more stable than another, or has improved stability, when it is moreresistant than the other enzyme to a loss of activity under the sameconditions, is more resistant to unfolding, or is more durable by anysuitable measure.

The term “host” or “host cell” means any cell of any organism that isselected, modified, transformed, grown, or used or manipulated in anyway, for the production of a substance by the cell, for example theexpression by the cell of a gene, a DNA or RNA sequence, a protein or anenzyme.

“DNA” (deoxyribonucleic acid) means any chain or sequence of thechemical building blocks adenine (A), guanine (G), cytosine (C) andthymine (T), called nucleotide bases, that are linked together on adeoxyribose sugar backbone. DNA can have one strand of nucleotide bases,or two complimentary strands which may form a double helix structure.“RNA” (ribonucleic acid) means any chain or sequence of the chemicalbuilding blocks adenine (A), guanine (G), cytosine (C) and uracil (U),called nucleotide bases, that are linked together on a ribose sugarbackbone. RNA typically has one strand of nucleotide bases.

A “polynucleotide” or “nucleotide sequence” is a series of nucleotidebases (also called “nucleotides”) in DNA and RNA, and means any chain oftwo or more nucleotides. A nucleotide sequence typically carries geneticinformation, including the information used by cellular machinery tomake proteins and enzymes. These terms include double or single strandedgenomic and cDNA, RNA, any synthetic and genetically manipulatedpolynucleotide, and both sense and anti-sense polynucleotide (althoughonly sense stands are being represented herein). This includes single-and double-stranded molecules, i.e. DNA-DNA, DNA-RNA and RNA-RNAhybrids, as well as “protein nucleic acids” (PNA) formed by conjugatingbases to an amino acid backbone. This also includes nucleic acidscontaining modified bases, for example thio-uracil, thio-guanine andfluoro-uracil.

Proteins and enzymes are made in the host cell using instructions in DNAand RNA, according to the genetic code. Generally, a DNA sequence havinginstructions for a particular protein or enzyme is “transcribed” into acorresponding sequence of RNA. The RNA sequence in turn is “translated”into the sequence of amino acids which form the protein or enzyme.

An “amino acid sequence” is any chain of two or more amino acids. Eachamino acid is represented in DNA or RNA by one or more triplets ofnucleotides. Each triplet forms a codon, corresponding to an amino acid.For example, the amino acid lysine (Lys) can be coded by the nucleotidetriplet or codon AAA or by the codon AAG. (The genetic code has someredundancy, also called degeneracy, meaning that most amino acids havemore than one corresponding codon.) Because the nucleotides in DNA andRNA sequences are read in groups of three for protein production, it isimportant to begin reading the sequence at the correct amino acid, sothat the correct triplets are read. The way that a nucleotide sequenceis grouped into codons is called the “reading frame.”

The term “gene”, also called a “structural gene” means a DNA sequencethat codes for or corresponds to a particular sequence of amino acidswhich comprise all or part of one or more proteins or enzymes, and mayor may not include regulatory DNA sequences, such as promoter sequences,which determine for example the conditions under which the gene isexpressed. Some genes, which are not structural genes, may betranscribed from DNA to RNA, but are not translated into an amino acidsequence. Other genes may function as regulators of structural genes oras regulators of DNA transcription.

A “coding sequence” or a sequence “encoding” a polypeptide, protein orenzyme is a nucleotide sequence that, when expressed, results in theproduction of that polypeptide, protein or enzyme, i.e., the nucleotidesequence encodes an amino acid sequence for that polypeptide, protein orenzyme.

The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing a protein by activating the cellular functions involved intranscription and translation of a corresponding gene or DNA sequence. ADNA sequence is expressed in or by a cell to form an “expressionproduct” such as a protein. The expression product itself, e.g. theresulting protein, may also be said to be “expressed” by the cell. Apolynucleotide or polypeptide is expressed recombinantly, for example,when it is expressed or produced in a foreign host cell under thecontrol of a foreign or native promoter, or in a native host cell underthe control of a foreign promoter.

The term “transformation” means the introduction of a “foreign” (i.e.extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, sothat the host cell will express the introduced gene or sequence toproduce a desired substance, typically a protein or enzyme coded by theintroduced gene or sequence. The introduced gene or sequence may also becalled a “cloned” or “foreign” gene or sequence, may include regulatoryor control sequences, such as start, stop, promoter, signal, secretion,or other sequences used by a cell's genetic machinery. The gene orsequence may include nonfunctional sequences or sequences with no knownfunction. A host cell that receives and expresses introduced DNA or RNAhas been “transformed” and is a “transformant” or a “clone.” The DNA orRNA introduced to a host cell can come from any source, including cellsof the same genus or species as the host cell, or cells of a differentgenus or species.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g. a foreign gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g. transcription and translation) of the introducedsequence.

Vectors typically comprise the DNA of a transmissible agent, into whichforeign DNA is inserted. A common way to insert one segment of DNA intoanother segment of DNA involves the use of enzymes called restrictionenzymes that cleave DNA at specific sites (specific groups ofnucleotides) called restriction sites. Generally, foreign DNA isinserted at one or more restriction sites of the vector DNA, and then iscarried by the vector into a host cell along with the transmissiblevector DNA. A segment or sequence of DNA having inserted or added DNA,such as an expression vector, can also be called a “DNA construct.”

A common type of vector is a “plasmid”, which generally is aself-contained molecule of double-stranded DNA, that can readily acceptadditional (foreign) DNA and which can readily introduced into asuitable host cell. A plasmid vector often contains coding DNA andpromoter DNA and has one or more restriction sites suitable forinserting foreign DNA. Promoter DNA and coding DNA may be from the samegene or from different genes, and may be from the same or differentorganisms. A large number of vectors, including plasmid and fungalvectors, have been described for replication and/or expression in avariety of eukaryotic and prokaryotic hosts. Non-limiting examplesinclude pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen,Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego,Calif.), or pMAL plasmids (New England Biolabs, Beverly, Mass.), andmany appropriate host cells, using methods disclosed or cited herein orotherwise known to those skilled in the relevant art. Recombinantcloning vectors will often include one or more replication systems forcloning or expression, one or more markers for selection in the host,e.g. antibiotic resistance, and one or more expression cassettes.Routine experimentation in biotechnology can be used to determine whichvectors are best suited for used with the invention. In general, thechoice of vector depends on the size of the polynucleotide sequence andthe host cell to be employed in the methods of this invention.

The term “expression system” means a host cell and compatible vectorunder suitable conditions, e.g. for the expression of a protein codedfor by foreign DNA carried by the vector and introduced to the hostcell. Common expression systems include bacteria (e.g. E. coli and B.subtilis) or yeast (e.g. S. cerevisiae) host cells and plasmid vectors,and insect host cells and Baculovirus vectors.

The term “in vivo mutagenesis” refers to a process of generating randommutations in any cloned DNA of interest which involves the propagationof the DNA in a strain of E. coli that carries mutations in one or moreof the DNA repair pathways. These “mutator” strains have a higher randommutation rate than that of a wild-type parent. Propagating the DNA inone of these strains will eventually generate random mutations withinthe DNA.

“Isolation” or “purification” of a polypeptide or enzyme refers to thederivation of the polypeptide by removing it from its originalenvironment (for example, from its natural environment if it isnaturally occurring, or form the host cell if it is produced byrecombinant DNA methods). Methods for polypeptide purification arewell-known in the art, including, without limitation, preparativedisc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phaseHPLC, gel filtration, ion exchange and partition chromatography, andcountercurrent distribution. For some purposes, it is preferable toproduce the polypeptide in a recombinant system in which the proteincontains an additional sequence tag that facilitates purification, suchas, but not limited to, a polyhistidine sequence. The polypeptide canthen be purified from a crude lysate of the host cell by chromatographyon an appropriate solid-phase matrix. Alternatively, antibodies producedagainst the protein or against peptides derived therefrom can be used aspurification reagents. Other purification methods are possible. Apurified polynucleotide or polypeptide may contain less than about 50%,preferably less than about 75%, and most preferably less than about 90%,of the cellular components with which it was originally associated. A“substantially pure” enzyme indicates the highest degree of purity whichcan be achieved using conventional purification techniques known in theart.

A “hydroxylation profile” of a product is a description of the numberand position of hydroxyl groups in the product. Thus, for example, analkane hydroxylase enzyme typically creates products having a definedhydroxylation profile, such that hydroxyl groups are placed at certainpositions on particular percentages of the final reaction products.Altering or modifying the hydroxylation profile of a product meanschanging the positions, or proportions, of hydroxyl groups in the finalreaction products. In another example, all of the products listed inTable 2 are used for the members of hydroxylation profile. For example,1-alcohol and 2-alcohols may make up the hydroxylation profile. Thus,Table 2 denotes the hydroxylation profiles of each of the mutants forsubstrates propane, butane, pentane, and octane, in this embodiment. A“variant” is distinguished from a mutant.

The general genetic engineering tools and techniques discussed here,including transformation and expression, the use of host cells, vectors,expression systems, etc., are well known in the art.

As used herein, a nucleic acid molecule is said to be “isolated” whenthe nucleic acid molecule is substantially separated from contaminantnucleic acid encoding other polypeptides from the source of nucleicacid.

Embodiments of the present invention further include fragments of anyone of the encoding nucleic acids molecules. As used herein, a fragmentof an encoding nucleic acid molecule refers to a small portion of theentire protein coding sequence. The size of the fragment will bedetermined by the intended use. For example, if the fragment is chosenso as to encode an active portion of the protein, the fragment will needto be large enough to encode the functional region(s) of the protein.For instance, fragments of the invention include fragments of DNAencoding mutant CYP153A6 proteins that maintain altered or enhancedregioselectivity for small-chain alkanes.

The determination of percent identity or homology between two sequencesis accomplished by BLAST (Basic Local Alignment Search Tool) analysisusing the algorithm of Karlin and Altschul, 1990, Proc. Nat'l Acad. Sci.USA, 87, 2264-2268, modified as in Karlin and Altschul, 1993, Proc.Nat'l Acad. Sci. USA, 90, 5873-5877. Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul, et al., 1990, J. Mol.Biol., 215, 403-410. BLAST nucleotide searches are performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches are performed with the XBLAST program, score=50, wordlength=3to obtain amino acid sequences homologous to the protein molecules ofthe invention. To obtain gapped alignments for comparison purposes,Gapped BLAST is utilized as described in Altschul, et al., 1997, NucleicAcids Res., 25, 3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.

The encoding nucleic acid molecules of the present invention may furtherbe modified so as to contain a detectable label for diagnostic and probepurposes. A variety of such labels is known in the art and can readilybe employed with the encoding molecules herein described. Suitablelabels include, but are not limited to, fluorescent-labeled,biotin-labeled, radio-labeled nucleotides and the like. A skilledartisan can employ any of the art known labels to obtain a labeledencoding nucleic acid molecule.

II. DIRECTED EVOLUTION GENERALLY

One technique to improve the alkane-oxidation capability of wild-typeCYP153A6 enzymes is directed evolution. General methods for generatinglibraries and isolating and identifying improved proteins according toone or more embodiments of the present invention using directedevolution are described briefly below. More extensive descriptions canbe found in, for example, Arnold, F., Accounts of Chemical Research,31(3): 125-131 (1998); U.S. Pat. No. 5,741,691; U.S. Pat. No. 5,811,238;U.S. Pat. No. 5,605,793; U.S. Pat. No. 5,830,721; and, InternationalApplications WO 98/42832, WO 95/22625, WO 97/20078, WO 95/41653 and WO98/27230, which are incorporated by reference herein.

The basic steps in directed evolution generally include: (1) thegeneration of mutant libraries of polynucleotides from a parent orwild-type sequence; (2) (optionally) expression of the mutantpolynucleotides to create a mutant polypeptide library; (3)screening/selecting the polynucleotide or polypeptide library for adesired property of a polynucleotide or polypeptide; and (4) selectingmutants which possess a higher level of the desired property; and (5)repeating steps (1) to (5) using the selected mutant(s) as parent(s)until one or more mutants displaying a sufficient level of the desiredactivity have been obtained. The property can be, but is not limited to,alkane oxidation capability and regiospecificity.

The parent protein or enzyme to be evolved can be a wild-type protein orenzyme, or a variant or mutant. The parent polynucleotide can beretrieved from any suitable commercial or non-commercial source. Theparent polynucleotide can correspond to a full-length gene or a partialgene, and may be of various lengths. Preferably the parentpolynucleotide is from 50 to 50,000 base pairs. It is contemplated thatentire vectors containing the nucleic acid encoding the parent proteinof interest may be used in the methods of this invention.

Any method can be used for generating mutations in the parentpolynucleotide sequence to provide a library of evolved polynucleotides,including error-prone polymerase chain reaction, cassette mutagenesis(in which the specific region optimized is replaced with a syntheticallymutagenized oligonucleotide), oligonucleotide-directed mutagenesis,parallel PCR (which uses a large number of different PCR reactions thatoccur in parallel in the same vessel, such that the product of onereaction primes the product of another reaction), random mutagenesis(e.g., by random fragmentation and reassembly of the fragments by mutualpriming); site-specific mutations (introduced into long sequences byrandom fragmentation of the template followed by reassembly of thefragments in the presence of mutagenic oligonucleotides); parallel PCR(e.g., recombination on a pool of DNA sequences); sexual PCR; andchemical mutagenesis (e.g., by sodium bisulfite, nitrous acid,hydroxylamine, hydrazine, formic acid, or by adding nitrosoguanidine,5-bromouracil, 2-aminopurine, and acridine to the PCR reaction in placeof the nucleotide precursor; or by adding intercalating agents such asproflavine, acriflavine, quinacrine); irradiation (X-rays or ultravioletlight, and/or subjecting the polynucleotide to propagation in a hostcell that is deficient in normal DNA damage repair function); or DNAshuffling (e.g., in vitro or in vivo homologous recombination of poolsof nucleic acid fragments or polynucleotides). Any one of thesetechniques can also be employed under low-fidelity polymerizationconditions to introduce a low level of point mutations randomly over along sequence, or to mutagenize a mixture of fragments of unknownsequence.

Once the evolved polynucleotide molecules are generated they can becloned into a suitable vector selected by the skilled artisan accordingto methods well known in the art. If a mixed population of the specificnucleic acid sequence is cloned into a vector it can be clonallyamplified by inserting each vector into a host cell and allowing thehost cell to amplify the vector and/or express the mutant or variantprotein or enzyme sequence. Any one of the well-known procedures forinserting expression vectors into a cell for expression of a givenpeptide or protein may be utilized. Suitable vectors include plasmidsand viruses, particularly those known to be compatible with host cellsthat express oxidation enzymes or oxygenases. E. coli is one exemplarypreferred host cell. Other exemplary cells include other bacterial cellssuch as Bacillus and Pseudomonas, archaebacteria, yeast cells such asSaccharomyces cerevisiae, insect cells and filamentous fungi such as anyspecies of Aspergillus cells. For some applications, plant, human,mammalian or other animal cells may be preferred. Suitable host cellsmay be transformed, transfected or infected as appropriate by anysuitable method including electroporation, CaCl₂ mediated DNA uptake,fungal infection, microinjection, microprojectile transformation, viralinfection, or other established methods.

The mixed population of polynucleotides or proteins may then be testedor screened to identify the recombinant polynucleotide or protein havinga higher level of the desired activity or property. Themutation/screening steps can then be repeated until the selectedmutant(s) display a sufficient level of the desired activity orproperty. Briefly, after the sufficient level has been achieved, eachselected protein or enzyme can be readily isolated and purified from theexpression system, or media, if secreted. It can then be subjected toassays designed to further test functional activity of the particularprotein or enzyme. Such experiments for various proteins are well knownin the art, and are described below and in the Examples below.

The evolved enzymes can be used in biocatalytic processes for, e.g.,alkane hydroxylation. The enzyme mutants can be used in biocatalyticprocesses for production of chemicals from hydrocarbons. Furthermore,the enzyme mutants can be used in live cells or in dead cells, or it canbe partially purified from the cells.

II. GROWTH of P. PUTIDA GPo12 (pGEC47ΔB) ON SHORT-CHAIN 1-ALKANOLS

In order to determine whether the strain P. putida GPo12 (pGEc47ΔB)could be used to select for improved terminal alkane hydroxylationactivity, its ability to grow on primary and secondary C₁ to C₈ alkanolswas measured. It was previously shown that strain P. putida GPo12(pGEc47ΔB) would grow on medium-chain length alkanols and on thecorresponding alkane only when complemented by a terminal alkanehydroxylase. (Smits, T. H., et. al., 2002, J. Bacteriol., 184,1733-1742). For these growth tests, cells from a Luria-Bertani brothpreculture were washed three times with M9 medium and used to inoculatethe 5-ml M9 main cultures in 14-ml tubes to an optical density (OD₆₀₀)of 0.1, then grown at 30° C. with continuous shaking.

No growth was observed on any of the secondary alcohols or methanolduring an 18 day period, but growth on ethanol, 1-propanol and 1-butanolwas comparable to the growth exhibited on the positive control grown onglucose, as depicted in FIG. 1. These results indicate that the terminalhydroxylation products of all short-chain n-alkanes except for methaneare readily utilized as carbon sources, while subterminal oxidationproducts are not. Thus, it was determined that strain P. putida GPo12(pGEc47ΔB) would be suitable for growth-based screening and selectionfor terminal hydroxylation of alkanes having various lengths.

III. DIRECTED EVOLUTION OF CYP153A6 VARIANTS

The recombinant host P. putida GPo12 (pGEc47ΔB) was engineeredspecifically for complementation studies with terminal alkanehydroxylases and was previously used to characterize members of the AlkBand CYP153 families. (Smits, T. H., et. al., 2002, J. Bacteriol., 184,1733-1742; van Beilen, J. B., et. al., 2006, Appl. Environ. Microbiol.,72, 59-65). This strain is a derivative of the natural isolate P. putidaGPo1 lacking its endogenous OCT plasmid, but containing cosmid pGEc47ΔB,which carries all genes comprising the alk machinery necessary foralkane utilization, with the exception of the deleted AlkB gene. Thisrecombinant host was complemented by a plasmid-encoded library of alkanehydroxylases and grown on various alkanes, resulting in an enrichment ofnovel, specific alkane-oxidizing terminal hydroxylases. Specifically,plasmid pCom8_cyp153A6 generated in Smits, T. H., et. al., 2001,Plasmid, 46, 16-24, were used with permission. Sequencing ofpCom8_cyp153A6 revealed that it was missing the first nine nucleotidesof the coding sequence of the monooxygenase gene compared to thepublished sequence. (Funhoff, E. G., et. al., 2006, J. Bacteriol., 188,5220-5227). Thus, the CYP153A6 parent enzyme (amino acid SEQ ID NO: 2)and its variant are three N-terminal amino acids shorter than thepublished sequence, but otherwise structurally identical and functional.

It was not efficient to use error-prone PCR to randomly mutate thetarget genes, as cloning of PCR products into the pCom vector resultedin fewer than 2,000 generated transformants. Therefore, mutant librarieswere constructed by complementing P. putida GPo12 (pGEc47ΔB) strainswith randomly mutated plasmids encoding CYP153A6. Mutator strains E.coli XL1-Red (Stratagene) and E. coli JS200 pEP polymerase I were usedto generate plasmid libraries according to the manufacturer's manual andCamps, M. and Loeb, L. A., 2003, Directed enzyme evolution: screeningand selection methods (F. H. Arnold and G. Georgiou, ed.), pp. 11-18,respectively. E. coli XL1-Red has deficiencies in the DNA repairmechanism that lead to a 5,000-fold increase in the general mutationrate. E. coli JS200 pEP Pol I expresses an engineered mutator DNApolymerase I which mainly amplifies plasmid DNA, with lower reliability,thus introducing mutations in the plasmid DNA. The nucleotide mutationlevel in XL1-Red after 2 weeks of continuous culturing was approximately0.1/kb, while four rounds of mutation in JS200 pEP Pol I yielded up to0.4/kb.

Cultures of both mutator strains were combined and the mutated plasmidswere transformed into P. putida GPo12 (pGEc47ΔB) through triparentalmating with the mediator strain E. coli CC118 (pRK600) and theappropriate E. coli DH5a donor. The growth selection was performed byculturing the resulting strain library in minimal medium with a specificalkane as the sole carbon source for up to 3 weeks. For more details andfurther discussion of the materials and methods used in the generationof the libraries, see Koch, Daniel J., et al., 2009, Appl. Environ.Microb., 75, 337-344, which is incorporated in its entirety by referenceherein.

The host, vector and operons of the best mutants were analyzedindividually by comparing them in growth tests to their wild-typecounterparts. For example, adapted vectors were isolated from thestrains and the CYP153A6 gene fdrA6 and fdxA6 operons were replaced withthe wild-type sequence by cloning before being mated into the wild-typehost. Additionally, potentially improved hydroxylase genes were reclonedinto a wild-type vector and transferred into a wild-type host.

A. CYP153A6 Variants

For P. putida GPo12 (pGEc47ΔB) complemented with CYP153A6 genes, it hadbeen observed that the host had to be adapted through prolongedcultivation on alkanes to obtain significant growth on alkanes, withoutany mutations occurring on the CYP153 genes themselves. Therefore, 21colonies from the first round of enrichment cultures were obtained tocompare to the parent strain in plate growth tests. Solid medium growthtests were chosen due to the instability of liquid minimal mediumcultures of P. putida GPo12 (pGEc47ΔB). Two strains adapted from P.putida GPo12 (pGEc47ΔB), Pcyp1 and Pcyp2, demonstrated faster growththan their respective parents on pentane. For the CYP153A6 system,adapted plasmid pCom8* enabled faster growth of P. putida GPo12(pGEc47ΔB) on pentane, even when it contained the wild-type operon.

In addition to creating these adapted hosts and plasmid, the first roundof in-vivo directed evolution generated enzyme variant CYP153A6-BMO1,which included a GCA to GTA change, resulting in the substitution A94Vin CYP153A6-BMO1. (This corresponds to an A97V substitution in onepublished sequence as the plasmid pCom8_CYP153A6 was missing its firstnine nucleotides when compared to the published sequence.) TheCYP153A6-BMO1 variant supported growth on butane, while the wild-typeCYP153A6 variant does not, as depicted in Table 1. Thus, the A94Vvariant enhances activity on smaller alkanes.

TABLE 1 Complementing Days required for growth to full lawn alkane withselected carbon source monooxygnease Ethane Propane Butane PentaneOctane CYP153A6 wild-type NG NG NG 2 2 CYP153A6-BMO1 NG NG 5 1.5 5 *NG =no growth detected during 3-week observation

While plasmid pCom8*_cyp153A6-BMO1 was subjected to a second round ofmutagenesis and mated into Pcyp1, there was no additional improvement inthe CYP153A6 system from CYP153A6-BMO1.

A schematic representation of the directed evolution process andresulting variant strains for CYP153A6 is depicted in FIG. 2.

IV. WHOLE CELL BIOCONVERSION

In order to quantify the effects of the mutations on enzyme performance,whole-cell bioconversions with the wild-type and mutant CYP153A6 enzymeswere performed using growth-arrested E. coli BL21(DE3) cells.

Whole-cell bioconversions with CYP153A6 enzymes were performed usinggrowth-arrested E. coli BL21(DE3) cells containing the CYP153A6 variantsexpressed from pCom8*. The typical concentration of folded CYP153A6 inthe cell suspensions used for bioconversion was 0.1 to 0.2 μM, withCYP153A6-BMO1 expressing about 20% less than its parent. Neither enzymeexhibited a significant decrease in apparent P450 concentration afterthe 60 minute bioconversion reactions. The bioconversion studydemonstrated significant altered activity and selectivity forCYP153A6-BMO1 that closely followed its growth complementationperformance. Conversion of butane yielded an average total concentrationof 393 uM 1-butanol in the aqueous phase after 60 minutes withCYP153A6-BMO1 versus 277 uM wild-type CYP153A6, as depicted in Table 2.

TABLE 2 Total hydroxylated product (μM)** Substrate* CYP153A6CYP153A6-BMO1 Propane 421 (59) 244 (68) Butane 356 (22) 439 (11) Pentane260 (62) 374 (49) Octane 78 (3)  69 (46) *Bioconversions with propaneand butane were performed in a bioreactor for 60 minutes with CYP153A6variants. Bioconversion mixtures with pentane and octane were shaken ina glass vial for 60 minutes for all enzymes. **Values in parentheses arethe percentage of 2-alkanol in the total hydroxylated product. Only 1-and 2- alkanols were formed in detectable amounts.

The average turnover rate of CYP153A6-BMO1 in 1-butanol productionincreased approximately 75% from the wild-type, from 28 min⁻¹ to 49min⁻¹. Surprisingly, the selectivity for terminal hydroxylation alsoincreased for CYP153A6-BMO1 when compared to wild-type CYP153A6, from78% to 89% of the total alkanols product, as shown in FIG. 3.

The A97V mutation in CYP153A6-BMO1 also improved activity andselectivity for conversion of pentane to 1-pentanol, but had theopposite effect with propane and octane.

CYP153A6-BMO1 is the first CYP153 enzyme shown to hydroxylate butane andenable its host to grow on this short-chain length alkane. Short-chain,gaseous alkanes are typically processed by specialized enzymes that areunrelated to the cytochrome P450s. Additionally, this is the first timethat biotransformations using CYP153 demonstrated activity on alkanessmaller than hexane. Consequently, CYP153A6-BMO1 was converted into agenuine butane monooxygenases by in-vivo directed evolution. Inaddition, the A97V mutation stabilized CYP153A6 and nearly doubled itshalf-life at 45° C.

EXAMPLES

One or more embodiments of the present invention are illustrated in thefollowing examples, which are provided by way of illustration and arenot intended to be limiting.

Example 1 Directed Evolution of CYP153A6-BMO1

Directed evolution of CYP153A6 was performed in vivo through themutagenesis of the pCom8 plasmid in E. coli XL1-Red (Stratagene)according to the manufacturer's manual and in E. coli JS200(pEP Pol I)as described in Camps, M. and Loeb, L. A., 2003, Directed enzymeevolution: screening and selection methods (F. H. Arnold and G.Georgiou, ed.), pp. 11-18. Mutated CYP153A6 gene fdrA6 and fdxA6 operonswere cloned into the original pCom8 plasmid as Kpn1-digested fragments.Restriction enzymes were obtained from Roche Molecular or New EnglandBiolabs.

Cultures of both mutator strains were combined and the mutated plasmidswere transformed into P. putida GPo12(pGEc47ΔB) through triparentalmating with the helper strain E. coli CC118 (pRK600) and the appropriateE. coli DH5a donor. Growth selection was performed by culturing theresulting strain library in minimal medium with an alkane as the solecarbon source for up to 3 weeks. More specifically, libraries of P.putida GPo12(pGEc47ΔB) were precultured on E2 minimal medium plates withantibiotics and 0.2% (wt/vol) citrate as the carbon source. Thelibraries were then enriched for improved strains through continuousgrowth in liquid E2 minimal medium with small-chain-length alkanes asthe sole carbon source. The liquid minimal medium cultures growing onalkanes were shaken in custom-made gas-tight flasks with 1% liquidalkane (pentane and octane) in a reservoir or in gas-tight serum bottles(Alltech).

Growth of the resulting adapted P. putida strains on minimal mediumplates with specific alkanes as the sole carbon source was measuredafter 3 weeks. Solid minimal medium growth tests on gaseous alkanes(Sigma-Aldrich) were conducted in gas-tight plastic containers (GasPak150 large anaerobic vented system; VWR), pressurized at 20 lb/in² for 20seconds (ethane and propane) or 10 lb/in² for 6 seconds (butane).

This example demonstrates one method by which evolution of the wild-typeCYP153A6 enzyme may occur. A mutant, CYP153A6-BMO1, was obtained afterenrichment and screening, as described above. FIG. 3 displays theregiospecific qualities of the CYP153A6-BMO1 mutant, which produced1-alkanols at a greater rate than wild-type CYP153A6—89% of totalalkanol product compared to 78%, respectively. As can be seen bycomparing CYP153A6-BMO1 to wild-type CYP153A6 in Table 2 and FIG. 3,there is an increase in production of butanol in the variant. Forexample, CYP153A6-BMO1 showed a 75% higher relative activity towards1-butanol production than wild-type CYP153A6. In terms of absoluteproduct concentration, CYP153A6-BMO1 produced 40% more 1-butanol thanwild-type CYP153A6 (390 μM versus 278 μM, respectively).

Sequencing of mutant CYP153A6-BMO1 revealed that it had a GCA to GTAnucleotide mutation in resulting in the amino acid substitutions A97V ofSEQ ID NO: 2. This corresponds to a A94V amino acid substitution in SEQID NO: 3.

Example 2 Bioconversions with CYP153A6-BMO1

E. coli BL21 (DE3) cells expressing CYP153A6 variants were preculturedin modified M9 medium with 1.5% yeast extract at 37° C. with shaking at250 rpm for 14 hours. Cultures with modified Mp medium with 1.5% yeastextract (120 ml) in 1,000 ml flasks were inoculated to an OD at 600 nm(OD₆₀₀) of 1.0 and grown at 37° C., 250 rpm, for 2.5 hours. The cultureswere then set to 25° C. at 200 rpm and induced with 0.4 mMdicyclopropylketone (Sigma Aldrich) after 30 minutes. The cultures werecentrifuged (10 minutes, 3,300×g, room temperature) 14 hours later andthe cell pellets resuspended in an equal volume of modified M9 mediumprepared without nitrogen. For cell dry weight determinations, 10-mlcell suspensions were washed once with distilled water and the cellpellets were dried for 3 days at 80° C. For bioconversion experimentswith liquid alkanes (i.e., pentane and octane), 250 μl of the alkane and20 mM glucose were added to a 1-ml suspension in a glass vial, capped,and incubated at a 60° angle at 25° C. and 200 rpm. After 60 minutes,reactions were stopped by addition of 200 μl of 1 N HCl.

Pentane bioconversion samples were then left open at 60° C. for 1 hourto allow the substrate to evaporate and subsequently pelleted, filtered,and subjected to gas chromatography analysis.

Octane bioconversion products were extracted by adding 250 μl hexane,vortexing for 30 seconds, and centrifuging at 14,000 rpm for 10 minutes.Alkanols were then detected in the top organic layer.

For bioconversion of gaseous alkanes, 80 ml of cell suspension and 15 μlof antifoam (Sigma-Aldrich) were stirred in a 100-ml bioreactor(Ochs-labor) at 25° C. Propane or butane were mixed in a 1:1 ratio withair, and the mixture was fed to the cells at an inlet gas flow rate ofapproximately 10 liter/hour. The reaction was started by addition of 20mM glucose and stopped after 60 minutes.

The results of the bioconversion experiments using CYP153A6 variants aredetailed in Table 2 and FIG. 3.

Example 3

CO-Binding Activity of CYP153A6-BMO1

Although pCom plasmids are not efficient expression platforms, CYP153A6expression was observed in E. coli, as indicated by the CO differencespectral peak at 450 nm in FIG. 4. In contrast, no 450 nm signal wasobserved in an empty pCom8* plasmid. Cytochrome P450s are typicallydifficult to express as it has been reported that the CO-bindingactivity of CYP153A6 is lost shortly after cell disruption at roomtemperature, even when the enzyme is isolated from its native host.However, CYP153A6 expressed well in E. coli DH5a and showed a stable COdifference spectrum for hours at room temperature if protease inhibitorwas added before cell disruption. The need of a protease inhibitor waseliminated when E. coli BL21 (DE3) was used to express CYP153A6.

E. coli BL21(DE3) cultures expressing CYP153A6 or CYP153A6-BMO1 wereconcentrated 5:1 in buffer. Cell extracts generated from these cultures,expressing wild-type CYP153A6 and CYP153A6-BMO1, retained fullCO-binding capacity for 24 hours when stored at 25° C. At 45° C.,CO-binding capacity decreased with time, showing a half-life of 638minutes for cell extract containing CYP153A6-BMO1 (diamonds) and 367minutes (squares) with wild-type CYP153A6, as depicted in FIG. 5. Theconcentration of properly folded P450 protein was determined from the COdifference spectrum of the 5×-concentrated cell extract aftersonification, removal of cell debris, and bubbling CO into the sample.Addition of a reducing agent to a concentrated cell extract was notnecessary and in fact decreased CO binding.

It is to be understood that the above-described arrangements are onlyillustrative of the application of the principles of the presentinvention and it is not intended to be exhaustive or limit the inventionto the precise form disclosed. Numerous modifications and alternativearrangements may be devised by those skilled in the art in light of theabove teachings without departing from the spirit and scope of thepresent invention. It is intended that the scope of the invention bedefined by the claims appended hereto.

In addition, the previously described versions of the present inventionhave many advantages, including but not limited to those describedabove. However, the invention does not require that all advantages andaspects be incorporated into every embodiment of the present invention.

All references cited herein, including but not limited to patents,patent applications, papers, text books, other publications, and thereferences cited therein, to the extent that they are not already, arehereby incorporated by reference in their entirety for all purposes.

1. An isolated mutant enzyme comprising the amino acid sequence of SEQID NO: 2, wherein the mutation consists of the A97V point mutation andthe mutant enzyme hydroxylates small-chain alkanes at rates greater thanthose of the wild-type enzyme.
 2. The isolated enzyme of claim 1,wherein said isolated enzyme can hydroxylate a small-chain alkanesubstrate at the terminal position.
 3. An isolated mutant enzymecomprising the amino acid sequence of SEQ ID NO: 3, wherein the mutationconsists of the A94V point mutation and wherein the enzyme hydroxylatessmall-chain alkanes at rates greater than those of the correspondingwild-type enzyme.
 4. The isolated enzyme of claim 2, wherein saidisolated mutant enzyme can hydroxylate a small-chain alkane substrate atthe terminal position.
 5. An isolated mutant enzyme having an amino acidsequence that has at least 90% sequence identity to SEQ ID NO:2, whereinthe mutation consists of the point mutation of A97V, and wherein themutant enzyme can hydroxylate an alkane at the terminal position.
 6. Theisolated enzyme of claim 5, wherein the amino acid sequence has at least95% sequence identity to SEQ ID NO:2 and the point mutation of A97V. 7.The isolated enzyme of claim 5, wherein the enzyme can regioselectivelyhydroxylate butane at the terminal position.
 8. An isolated mutantenzyme having an amino acid sequence that has at least 90% sequenceidentity to SEQ ID NO:3, wherein the mutation consists of the pointmutation of A94V, and wherein the mutant enzyme can hydroxylate asmall-chain alkane at the terminal position.
 9. The isolated enzyme ofclaim 8, wherein the amino acid sequence has at least 95% sequenceidentity to SEQ ID NO:3 and the point mutation of A94V.
 10. A method ofmaking a mutant CYP153A6 enzyme having an altered ability to hydroxylatea small-chain alkane substrate at the terminal position, comprising: (a)creating a CYP153A6 enzyme mutant library, wherein the CYP153A6 enzymethat is mutated has the amino acid sequence of SEQ ID NO:2 or SEQ IDNO:3; (b) providing a host microbial cell culture capable of growing ona 1-alkanol; (c) transforming said host cell culture with said mutantlibrary; (d) growing said host cell culture on an alkane substrate; and,(e) selecting and isolating the mutant enzymes based on the ability tohydroxylate a small-chain alkane at the terminal position.
 11. Themethod of claim 10, wherein the substrate is butane.